Encapsulated reagents and methods of use

ABSTRACT

The present invention contemplates use of encapsulated aqueous and non-aqueous reagents, solutions and solvents and their use in laboratory procedures. These encapsulated aqueous or non-aqueous reagents, solutions and solvents can be completely contained or encapsulated in microcapsules or nanocapsules that can be added to an aqueous or non-aqueous carrier solution or liquid required for medical and research laboratory testing of biological or non-biological specimens.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/549,286, filed Aug. 23, 2019, which is a continuation of U.S.application Ser. No. 14/673,409, filed Mar. 30, 2015, now U.S. Pat. No.10,393,632, which is a continuation of U.S. application Ser. No.14/336,500, filed Jul. 21, 2014, now U.S. Pat. No. 8,993,235, which is acontinuation of U.S. application Ser. No. 13/944,619, filed Jul. 17,2013, now U.S. Pat. No. 8,785,124, which is a continuation of U.S. Ser.No. 13/144,854, filed Jul. 15, 2011, now abandoned, which claimspriority under 35 U.S.C. 371 of International ApplicationPCT/US10/21200, filed Jan. 15, 2010, which claims the benefit of U.S.Provisional Application Ser. No. 61/145,269, filed Jan. 16, 2009. Theentire contents of each are hereby expressly incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

In a microscope slide treatment method known in the prior art, a testspecimen which is attached to a microscope slide is treated using twophase-separating liquids. In this method, first, an aqueous reagent isplaced on an upper surface of the microscope slide to which the specimenis attached. Then a layer of mineral oil or other immiscible oil isplaced over the aqueous reagent. The two different phases remainseparated even after stirring or agitation. This is desired in thisexample, however, because the purpose of the placement of the oil layerover the aqueous reagent is to reduce the evaporation of the aqueousreagent during the timed incubation steps (e.g., heating). However, thismethod requires two separate steps to dispose the reagent and oil on theslide. For example, in one alternative, the aqueous reagent is placedover the biological specimen first and then, in a second step, the oillayer is placed over the aqueous reagent. Alternatively, one couldenvision first placing the oil layer over the biological specimen, andthen placing the aqueous reagent onto the oil layer thereby wherein theaqueous reagent then submerges through the oil layer to the microscopeslide surface whereby the oil layer floats on top of the aqueousreagent. A significant disadvantage of this method is that the aqueouslayer tends to remain localized at the specific location where theaqueous reagent was first placed on the slide, once the oil layer isplaced thereon. If the aqueous reagent is placed on top of the oil layerso the aqueous reagent layer passes through the oil layer but theaqueous reagent layer partially or entirely misses the specimen by notcovering the whole specimen area, the aqueous reagent layer is fixed inthat exact position once it passes through the oil layer and thus thespecimen is not treated with the aqueous reagent. If one were to place,for example, a stir stick or stir device through the oil layer and downto the aqueous layer to mix or move the aqueous layer, the aqueousreagent tends to remain in its original location of placement and cannotbe moved to a more useful or appropriate area upon or around the slideor specimen. This reduces the ability of the specimen to react with thereagent in this method. A solution to this problem to increase theefficiency of the process and to minimize the chances of damaging thespecimen is desirable.

DETAILED DESCRIPTION OF THE INVENTION

The present invention contemplates use of encapsulated aqueous andnon-aqueous reagents, solutions and solvents and their use in laboratoryprocedures. These encapsulated aqueous or non-aqueous reagents,solutions and solvents can be completely contained or encapsulated inmicrocapsules or nanocapsules that can be added to an aqueous ornon-aqueous carrier solution or liquid required for medical and researchlaboratory testing of biological or non-biological specimens (alsoreferred to herein as “testing” or “test” specimens). Where used hereinthe term “encapsulated reagent” is intended to refer also to“microencapsulated reagents” and/or to “nanoencapsulated reagents”.Further, where used herein, the term “capsule” is intended to refer to“microcapsules” or “nanocapsules”.

Where used herein the term “biological specimen” includes, but is notlimited to, unprocessed specimens, processed specimens, paraffinembedded tissue, whole mounts, frozen sections, cell preps, cellsuspensions, touch preps, thin preps, cytospins, and other biologicalmaterials or molecules including blood, urine, cerebrospinal fluids,pleural fluids, ascites fluids, biopsy materials, fine needle aspirates,pap smears, swabbed cells or tissues, microbiological preps includingbacteria, viruses, parasites, protozoans, biochemicals including, butnot limited to proteins, DNA, RNA, carbohydrates, lipids, ELISA reagentsand analytes, synthetic macromolecules, phospholipids, supportstructures of biological molecules (e.g., metals, beads, plastics,polymers, glass), or any other materials attached to a biologicaltesting substrate for processing, examination, or observation.

The capsules of the present invention are generally considered to havediameters in the range of from less than 0.001 angstrom (0.0001 nm) to3000 microns (3000 μm). Preferably the range is from 1 angstrom (0.1 nm)to 1000 micrometers. The range can also be from 1 nanometer to 1000microns. The shells or encapsulating material that make up themicrocapsules or nanocapsules can be gelatin, polyvinyl alcohol, urea,melamine formaldehyde polymers, acrylics, urethanes, vinyl acetatecopolymers, oily, lipid, or non-aqueous soluble materials and polymers,water, or aqueous based materials and polymers. The encapsulationprocesses used to form the encapsulated reagents include, but are notlimited to, coacervation, vapor deposition, fluid bed coating,entrapment/matrix, macro-emulsion, mini-emulsion, micro-emulsion,micro-encapsulation techniques, macro-encapsulation techniques,dispersion polymerization, in situ polymerization, liposomal, alginateencapsulation, solvent phase separation, and pan coating. The micro- ornanoencapsulated reagent product can be delivered as a dry, free-flowingpowder, as a slurry, or in the form of wet filter cake.

Reagents and compounds which may be microencapsulated ornanoencapsulated as contemplated for use in the present inventioninclude, by way of example only, not by way of limitation, Dextransulfate, formamide, SSC (sodium chloride sodium citrate solutions), DIwater, Millipore™ water, RNAase-free and DNAase-free water, DAPI counterstain, propidium iodine counterstain, counterstains, salts, buffers,chemicals, DNA probes, RNA probes, protein probes, antibodies,monoclonal antibodies, polyclonal antibodies, probes, detectionreagents, stains, biological stains, dyes, washes, rinses, enzymes,antigen retrieval solutions or buffers, ionic, non-ionic, anionic,cationic, neutral detergents and surfactants, thermoplastics, mountants,oils, lipids, phospholipids, molecular biological building blocks,carbohydrates, sugars, lyophilized or desiccated powder or dry reagentsthat can be reconstituted with and aqueous or non-aqueous solution,preservatives, cover slip media, liquified thermoplastic cover slipmedias, xylene, toluene, acetone, petroleum distillates, ferrofluids,magnetic particles in a fluid, colloidal gold conjugated reagents,iron-containing fluids, iron particles, magnetic particles, organicsolvents, inorganic solvents, aqueous solvents, non-aqueous solvents,lipid based solvents, emulsions, liquid chemicals, Histology clearingreagents, Histology deparaffinizing reagents, Histology hydratingreagents, Histology dehydrating reagents, Histology fixatives,formaldehyde, alcohols, polyols, magnetic particle powders, powders,lyophilized reagents, lyophilized antibodies, lyophilized molecularprobes like RNA and DNA, dry chemicals, dry, powdered, or lyophilizedstains and reagents, fluorescent conjugated reagents like antibodies,stains, and molecular probes, and detection reagents, chromogens, DAB,hydrogen peroxide, naphthol phosphate, fast red chromogen, acids, bases,HCL, formic acid, glacial acetic acid, sodium hydroxide, potassiumhydroxide, aqueous and non aqueous liquids, and any other reagent and/orchemicals including liquids, dry reagents, desiccated reagents, gelreagents, colloidal reagents, emulsions reagents and any other reagentor chemical known in the art of medical and research laboratory testingreagents or chemicals. These reagents will be referred to herein as“encapsulated reagents”. The solutions or liquids to which theseencapsulated reagents can be added may be referred to elsewhere hereinas “solutions” or more particularly as “carrier solutions”. The above isexemplary only and is not intended to be an exhaustive list of thereagents or compounds which may be encapsulated or used herein.

Examples contemplated herein of the use of these encapsulated reagentsinclude for example (1) addition of aqueous-based encapsulated reagentsto non-aqueous based solutions, (2) addition of non-aqueous basedencapsulated reagents to aqueous-based solutions, (3) addition ofaqueous-based encapsulated reagents to aqueous-based solutions, (4)addition of non-aqueous based encapsulated reagents to non-aqueous basedencapsulated solutions, and (5) addition of both aqueous-based andnon-aqueous based encapsulated reagents to either an aqueous- ornon-aqueous solution, and wherein the resulting combination solutionscontain such encapsulated reagents in a homogenous, soluble, orcolloidal, emulsions, or at least partially soluble liquid mixture.

It is known that when adding a typical aqueous-based reagent to anon-aqueous solution, or vice-versa, the two different phases separate.There is therefore a need to be able to mix aqueous-based reagents withnon-aqueous-based solutions, and non-aqueous-based reagents withaqueous-based solutions to form homogenous solutions of both the reagentand the solution without the usual phase separation of both.Encapsulation of the reagent as contemplated herein enables theformation of such homogenous mixtures.

The problem in the prior art method described above in the Backgroundmay be addressed by using a three-step procedure involving firstdisposing an aqueous detergent-containing layer over the entire area ofthe slide (or analytic substrate, as defined herein) where one wouldlike the aqueous reagent layer to be positioned once the oil layer isadded, or vice versa. In this method, an aqueous detergent-containinglayer can be placed first on the microscope slide, followed by additionof the aqueous reagent layer, and then followed by addition of the oillayer in a third subsequent step. In the presence of these three layers,the aqueous reagent layer can be moved or mixed on the slide anywherethe aqueous detergent layer is present. However, this method is highlyinefficient in both time, materials, and cost required to perform astaining protocol which requires an oil layer or liquid phase/separationprotocol. A further disadvantage of this three-step method is that theaqueous detergent layer must always be added first, though either theaqueous reagent layer, or the oil layer can be added next. Still,whether the oil layer is added, second or third following addition ofthe aqueous reagent layer, it is obvious that the method is stillrequires three separate steps and is a costly time and materialconsuming protocol.

The present invention provides a solution to the problems of the priorart method. In the present invention, one or more reagents which areencapsulated, by microencapsulation and/or nanoencapsulation, are addedto aqueous or non-aqueous solutions for use as single ready-to-usesolutions for laboratory testing of specimens. The physical and/orchemical makeup of the encapsulated reagents is such that themicrocapsule or nanocapsule containing the reagent is soluble, at leastpartial soluble, or colloidal in a solution which has a different liquidphase or density than that of the encapsulated reagent. In analternative embodiment, the encapsulated reagents have the same orsimilar densities or liquid phase of the solution. Further, themicrocapsule or nanocapsule could have the same or similar density orthe same or similar liquid phase of the solution regardless if thereagent encapsulated therein has the same or similar liquid phase ordensity. It is an object of the present invention to encapsulatereagents wherein the chemical and/or physical properties of theencapsulating material (the outer shell of the capsule) are like orsimilar to that of the solution to which the encapsulated reagent willbe added thereby allowing the capsules to be soluble, at least partiallysoluble, or colloidal in or with the solution. The reagent, onceencapsulated, therefore would be soluble, at least partially soluble, orcolloidal in relation to the solution. In a preferred embodiment of thepresent invention, a solution comprising at least one encapsulatedreagent which has a density different from the solution, is provided andapplied to a specimen. In an alternative embodiment of the presentinvention, a solution is provided which has at least one encapsulatedreagent having a density which is the same as or similar to the solutioncontaining it, then the solution is applied to a specimen.

The analytic substrates used in the present invention may be constructedof glass, plastic, synthetic polymers, ceramics, or metals and may be ofany size or shape known in the art of laboratory examination, forexample including any laboratory support structure or testing structureor device used in laboratory testing or examination including, but notlimited to, microscope analytic plates, analytic substrates, medical andresearch laboratory testing substrates, diagnostic substrates,biological testing substrates, substrates, microscope slides, testtubes, Petri dishes, micro arrays, biochips, testing plates, containers,beads, and testing strips and any other natural or synthetic substrateor device used in the art for medical, research, laboratory, anddiagnostic testing, in-vitro testing and/or analysis of at least onebiological specimen.

The process wherein the microcapsule or nanocapsule opens ordisintegrates upon the analytic substrate to release the reagentcontained therein is referred to herein as “disruption”. Disruption,once started, can be immediate (i.e., “immediate release”), or can be aslow or gradual release (i.e., “controlled” or “timed” release). Thetype of disruption necessary for the capsule to release its contents isreferred to herein as the “disruption mode”. The causes or stimuli ofthe disruption mode can be, for example, temperature changes ordifferentials, high temperature (e.g., 150° C.-200° C.), mediumtemperature (e.g., 100° C.-150° C.), low temperature (e.g., 25° C.-100°C.), heat, mechanical disruption, e.g., by agitation, sonic disruption,magnetic disruption, electric disruption, microwave disruption, UVlight, infrared light, laser light, light, other types ofelectromagnetic radiation or energy, pH changes, pressure differentials,high pressure, medium pressure, low pressure, pressure changes above orbelow atmospheric (e.g., pressures of 1 psig-5000 psig; 1-10 psig; 10-50psig; 50-100 psig; 100-150 psig; 150-200 psig; 200-300 psig; 300-500psig; or 500-5000 psig), vacuum, and vacuum changes, time release, timedependent, chemical reactions, chemical changes, and physical reactions,and physical changes. Various disruption modes can be combined, e.g.,temperature and pressure; pH and heat; time and pressure; and pressureand time, for example.

In an alternate embodiment, the “disruption” of the microcapsule ornanocapsules contained within a carrier solution occurs upon thecombination of the carrier solution with at least one other solution orcompound. The carrier solution in this embodiment has at least onereagent present in a microcapsule or nanocapsule, and the secondsolution optionally has at least one microcapsule or nanocapsule presentwhich encapsulates a reagent. If the second solution doesn't have anyencapsulated reagent present, the chemical activity, physical activity,or reaction when combining with the second solution can initiatedisruption of the microcapsule or nanocapsule in the carrier solution.The combination of at least two solutions (carrier and second solution)each having at least one reagent present in a microcapsule ornanocapsule or only one of the two solutions having encapsulated reagentpresent when mixed can, in alternate embodiment, now “activate”(disrupt) the microcapsule(s) or nanocapsule(s) in the combinedsolutions. Activation of the microcapsule or nanocapsule is intended tomean the ability for the microcapsule or nanocapsule to become unstableor disruptable only after the at least two solutions are combined,wherein if the solutions are not combined, the microcapsule ornanocapsule are stable against disruption modes when they are in theirseparate solutions. Only when the at least two solution are mixedtogether are the microcapsule or nanocapsule disruptable in thisembodiment. In an alternative embodiment, there can be one or moresolutions combined to “activate” any microcapsule or nanocapsule presentin at least one of the solutions of the combination.

A solution of the present invention may comprise an encapsulated reagentwherein the capsule is responsive to a single type of disruption mode,e.g., a pressure or sonic sensitive encapsulation, or a single solutionmay comprise a plurality of encapsulated reagents each wherein each typeof capsule is responsive to a different type of disruption mode. In oneembodiment, for example, a solution could contain three types ofencapsulated reagents, each used in one of three different steps of atest protocol. For example, the first encapsulated reagent having acapsule with a “heating” disruption mode could be released to react withthe slide specimen when the slide is heated. The second and thirdencapsulated reagents could have capsules having disruption modes whichwere not activated by or affected by heat but which were activated by apH change or pressure change, for example, or other condition describedherein.

As explained herein, embodiments of the present invention includecarrier solutions which comprise only a single type of solute andcarrier solutions which comprise multiple (two or more) solutes. In oneembodiment of the present invention, the carrier solution can be mixedwith another solution or solutions absent encapsulated reagent(s) orwith encapsulated reagents, wherein the mixing of the two solutions maycause disruption of a encapsulated reagent in one, both, or all of thesolutions, or the mixing of the two solutions does not disrupt theencapsulated reagent(s) but rather the mixed solutions remain inassociation with each other as a homogenous mixture, colloidal mixture,emulsion solution, phase separated solution, suspension solution,miscible solution, or immiscible solution, which the encapsulatedreagents remain in an intact encapsulated condition. A list of reagentswhich may be encapsulated in accordance with the present invention isprovided above. This list is exemplary only and does not constitute anylimitation of the possible combinations of encapsulated reagents andreagents or solutes present in the one or more carrier solutions.

In one embodiment, the microencapsulated or nanoencapsulated reagentscould be manufactured by Particle Sciences, Inc. 3894 Courtney Street,Bethlehem, Pa. 18017-8920 US and/or by Microtek Laboratories, Inc. 2400East River Road, Dayton, Ohio 45439.

In various embodiments of the invention, exemplary methods of producingthe microcapsules and nanocapsules used herein and descriptions of thecapsular “shells” include, but are not limited to, those disclosed inthe following U.S. patents and Published patent applications, all ofwhich are hereby expressly incorporated by reference herein in theirentireties. U.S. patents include, but are not limited to, U.S. Pat. Nos.7,588,703, 7,462,365, 7,270,851, 7,052,766, 6,989,196, 6,932,984,6,913,767, 6,881,482, 6,828,025, 6,777,002, 6,767,637, 6,716,450,6,599,627, 6,555,525, 6,465,425, 6,458,118, 6,265,389, 6,214,300,6,146,665, 6,113,935, 6,103,271, 6,080,412, 5,925,464, 5,863,862,5,766,637, 5,650,102, 5,643,605, 5,552,149, 5,540,927, 5,508,041,5,503,851, 5,503,781, 5,464,932, 5,418,010, 5,407,609, 5,403,578,5,362,424, 5,277,979, 5,204,184, 5,164,126, 5,164,096, 5,160,529,5,100,673, 5,091,122, 5,066,436, 5,051,306, 4,942,129, 4,895,725,4,803,168, 4,766,012, 4,764,317, 4,711,783, 4,675,189, 4,673,595,4,594,370, 4,521,352, 4,518,547, 4,508,760, 4,389,330, 4,269,729,4,211,668, 4,193,889, and 4,123,382. U.S. Published patent applicationsinclude, but are not limited to, 2009/0311329, 2009/0253901,2009/0214633, 2009/0202652, 2009/0104275, 2009/0098628, 2009/0047314,2008/0234406, 2008/0138420, 2008/0102132, 2008/0031962, 2007/0077308,2007/0027085, 2007/0009668, 2006/0237865, 2006/0188464, 2006/0127667,2006/0093808, 2006/0071357, 2006/0051425, 2004/0228833, 2004/0065969,2004/0032038, 2003/0138491, 2003/0062641, 2002/0160109, and2002/0064557.

Examples of reagent (e.g., DNA, RNA, ISH, FISH) reacting with thebiological specimen.

As noted above, the encapsulated reagent could be of a different phaseor density than that of the solution within which the encapsulatedreagent is to be disposed. For example, a non-aqueous solution, such asan oil-based solution, could comprise a soluble, partially soluble, orcolloidal suspension, of one or more encapsulated aqueous reagents forperforming in situ hybridization of a DNA or RNA probe to a target DNAor RNA present in a specimen on a slide or other substrate. For example,the specimen is present on a microscope slide and the slide is heated to70°-110° C. The oil solution with its encapsulated reagents presenttherein is added to the microscope slide. Heating at a temperature of72° C., for example would cause the disruption of the capsule of theencapsulated reagent, for example, and the aqueous reagents thereinwould thereby be released into the oil solution. The aqueous reagentsquickly separate away from the oil layer and are deposited onto themicroscope slide and onto specimen thereon. Wherever the oil solution ispresent on the microscope slide, there would now be a layer of aqueousreagents that had separated from the oil solution and had migrated tothe surface of the microscope slide. Present on the surface of themicroscope slide therefore, would be an aqueous reagent layer, with theoil layer of the original solution over the aqueous reagent layer. Theaqueous layer could then be agitated or stirred about the slide becausethe encapsulated reagent preferably had present as one of the reagentstherein a detergent for enhancing the dispersion and distribution of theaqueous reagents under the oil layer and upon the slide surface andspecimen thereon.

In an alternative embodiment, the microscope slide, with biologicalspecimen attached thereto, is first flooded with a wash buffer that hasat least one detergent and/or surfactant present. Excess wash buffer isremoved to leave only a residual layer of wash buffer on the microscopeslide and biological specimen (e.g., approximately 1-100 micro liters ofwash buffer remaining on the slide). An oil-based carrier solutioncontaining the encapsulated aqueous reagent is now added to the wetslide. The capsules of encapsulated aqueous reagent are disrupted andthe aqueous reagent therein is released and deposited onto the residualwash buffer. The aqueous reagent moves to the biological specimen andsurrounding areas of the microscope slide. The oil layer is above theaqueous reagent. The aqueous reagent can be moved on the microscopeslide and biological specimen wherever the residual wash buffer islocated by agitating the aqueous reagent or moving the oil layer whichin turn would move the aqueous reagent about the biological specimenand/or microscope slide.

It is contemplated in the present invention that each reagent used in anin-situ hybridization process or other treatment methods contemplatedherein can be encapsulated separately, or as mixtures that areencapsulated together. The reagents necessary for a denaturing andhybridization step of the in-situ hybridization protocol can be, forexample, formamide, dextran sulfate, DI water, detergents, and therequired DNA or RNA probe for example. This single solution comprisingreagent capsules dispersed therein can be used for the steps ofdenaturing and hybridization of the target nucleic acid specimen is anovel one step solution which features all the advantages of use of anoil layer to inhibit evaporation. The use of such a single solution ofthe present invention is novel because in the prior art process ofco-denaturing a nucleic acid with heat to denature and hybridize anucleic acid to the target DNA present in a specimen attached to amicroscope slide, one would necessarily have to use a slide having asealed cover slip sealed with an adhesive over the specimen with thenucleic probe mixture placed underneath the sealed cover slip. In themethod of the prior art, after heating, usually at 72° C. for 10minutes, for example, the attached cover slip then must be removed whichcan cause damage to the specimen. Alternatively, the novel method of thepresent invention, wherein a single solution that has all the necessaryreagents for hybridization present in microcapsules or nanocapsules isused, eliminates all the multiple steps of having to add differentreagents and the manual steps of preparation during an in-situhybridization. The single solution method of the present invention hasall the necessary reagents, present within capsules in the solution, sothere is no waste of reagents such as of very expensive nucleic acidprobes. The single solution of the present invention may be added to thespecimen on the microscope slide or other biological container orsubstrate. During the co-denaturing step in the present invention, theheat (or other disrupting mode contemplated herein) disrupts themicrocapsules or nanocapsules thereby causing release of the reagentsonto the specimen. There is no need for a cover slip to cover thespecimen and reagents during the hybridization process because thesingle solution may be oil based while the reagents encapsulated may beaqueous-based. The oil phase in the solution will separate from theaqueous phase comprising the released reagents, thereby forming anevaporation barrier over the aqueous reagents thereby inhibiting theevaporation of the aqueous reagents during the heating or denaturing ofthe DNA or RNA of the target specimen. This single oil-based solutioncontaining the encapsulated aqueous reagents can be applied by a dropperbottle, pipette, or any other type of dispenser including the dispenserdescribed below and those noted for example in U.S. publishedapplications 2006/0281116, 2006/0275889, and 2006/0275861, each of whichis expressly incorporated herein by reference.

In a preferred embodiment, the encapsulated reagent is also very stableand has a long shelf life of greater than one year at room temperatureor under 2-4° C. refrigeration vs. the non-encapsulated reagent thatwould normally require freezer storage at −20° C. or ultra cold freezerstorage below 0° C. to maintain its freshness and viability. Theencapsulation protects the temperature sensitive reagent and thereagent, as encapsulated by the methods of the present invention has avery long shelf life at normal refrigeration (2-4° C.) or at roomtemperature (25-30° C.). This stability provided by encapsulating thetemperature-sensitive reagents is advantageous during the packing andshipping of these reagents. Many of the conventional, non-encapsulated,reagents listed herein must be shipped via “Next Day Air” in a containerthat is cooled by cold packs or dry ice. The encapsulated temperaturesensitive reagents of the present invention can instead be shippedground in a normal shipping container which is more cost effectivewithout sacrificing freshness or degradation due to insufficientpacking, insufficient temperature controlled shipping environments, andincreasingly high shipping fees of next day air shipping requirements ofthe non-encapsulated temperature sensitive reagents. The encapsulationof the present invention inhibits the degradation of these proteins(e.g., antibodies) and chemical from bacterial or fungal attack ordegradation. The capsules preferably have anti-fungal and anti-bacterialproperties to inhibit the degradation of these proteins and chemicalswhen stored at room temperature or at refrigerated conditions and duringshipping and use.

One embodiment of the invention is directed to a novel deparaffinizingsolution. In this embodiment, one or more deparaffinizing reagents suchas, but not limited to, like xylene, petroleum distillates, or othernon-aqueous deparaffinizing solutions and solvents have dispersedtherein an encapsulated reagent such as (but not limited to) an alcoholor other water soluble substance. This deparaffinizing solution can beplaced onto the paraffin embedded tissue section thereby causing theparaffin associated with the biological specimen to soften and dissolveinto the deparaffinizing reagent thereby removing the paraffin from themicroscope slide and the biological specimen. Following dissolution ofthe paraffin, the microscope slide and the biological specimen must nowbe rinsed with a chemical solution that is miscible with thedeparaffinizing solution. In the present invention, this “rinsing”solution or reagent is contained within the capsules contained withinthe deparaffinizing solution. If the deparaffinizing solution was xylenefor example, the encapsulated rinse agent therein could be a reagentgrade alcohol, for example. This alcohol is miscible with the xylenedeparaffinizing solution. The capsules would then be disrupted byexposure of xylene/paraffin mixture to a predetermined disruptioncondition such as described elsewhere herein causing release of thealcohol into the xylene/paraffin mixture. The alcohol/xylene/paraffinmixture would then rinsed or removed from the slide (or analyticsubstrate) and biological specimen by an aqueous reagent (such as, butnot limited to, water) which is miscible with thealcohol/xylene/paraffin mixture. The alcohol in this one-stepdeparaffinizing solution is released from the microencapsules by acondition such as time, heat, pressure, vacuum, mechanical disruption orcombinations thereof, or other conditions described herein. This releaseof the alcohol into the deparaffinizing solution makes analcohol/xylene/paraffin liquid that can easily be rinsed by an aqueousrinse buffer. The advantage of this deparaffinizing solution whichcontains the encapsulated alcohol is that there are fewer stepsnecessary for deparaffinization and the use of minimal reagents that canbe used effectively and efficiently with less waste and reduced disposalcost when dealing with hazardous deparaffinizing solutions like xylene.Although the example of deparaffinization provided above describes theuse of xylene and alcohol, other deparaffinizing solutions are known andcontemplated for use instead. For example, deparaffinizing solutionsthat are aqueous-based can be used with the above-mentioned example ofthe present invention. Any deparaffinizing solution, whether aqueous ornon-aqueous based, can be used as long as there is at least onemicroencapsulated reagent present in the deparaffinizing solution thatis soluble, miscible, colloidal, or at least partially soluble in thedeparaffinizing solution to prepare the paraffin/deparaffinizingsolution to be rinsed with an aqueous buffer. An example of anaqueous-based deparaffinizing solution is the use of detergents in wateror in water and solvent compositions (i.e., polar and non-polar solventswith detergents in water). The deparaffinizing carrier solution whetheraqueous based or non-aqueous based can have in the alcohol-containingcapsule an additive, such as a surfactant, detergent, polyols, or othercomponent. Or in an alternate embodiment, these additives can beencapsulated in their own microcapsules or nano-capsules. The reagentsmay be encapsulated by any appropriate means contemplated in any of thedescription, patents, or published applications described herein.

The encapsulation material used to form the microcapsules ornanocapsules of the present invention, in an alternate embodiment, canalso have magnetic properties and/or electrical properties.Alternatively, the encapsulation material can be acted on by a magneticcurrent or electrical current present within or adjacent the solutionfor use with the magnets described elsewhere herein. These magneticand/or electrical properties of the capsules may be advantageous when itbecomes necessary to mix, agitate, align, or otherwise move the capsulesin relation to the solution or the specimen. For example, capsules thathave magnetic properties can be moved toward or away from the specimenby magnets associated with the apparatus upon which the slide is placed,such as the instruments described in the published U.S. patentapplications noted above. The magnets can, for example, move certaincapsules toward the specimen just before disruption of the capsules tohave the reagents as close to the specimen as possible to reduce thetime of incubation and efficiency of the binding activity of the reagentwith the specimen. Further, in other embodiments, the capsules can havea net charge such that they can be acted on by electrical currents to bepulled toward or repelled from the specimen or solution or otherlocation on the microscope slide. For example, an electrical current maypass through the solution thereby pulling the charged capsules towardthe specimen just prior to disruption. Alternatively, the polarity ofthe electrical current could be reversed to move the expelled ordisrupted capsules out of the way of a different capsule containing adifferent reagent moving toward the specimen for the next subsequentreagent step. The electrical and/or magnetic properties of the capsulehave further advantages of enabling the capsules to be mobile or to movein the solution when an electrical field or magnetic field is generatedabout the solution for mixing the capsules, or mixing the reagent orreagents disrupted from the capsules, for example, or by moving thedisrupted capsules thereby producing a swirling, moving, or agitationmotion in the solution or its respective liquid phase to mix or agitatereagents in relation to the specimen. The capsules can be moved, or theremains of the disrupted capsules can be moved, by a magnetic field orelectrical field or both, to agitate, move, or mobilize othernon-disrupted capsules, expelled reagents, and or the solution.

In another embodiment of the present invention, the capsules may havemagnetic or electrical properties present on or within each capsule inthe solution which can be used to evenly distribute or dispense thecapsules in the solution. Each capsule or groups of similar capsulescould have a positive or negative net charge present therein to repelother capsules in the solution to enhance the distribution or dispersionof different capsules within the solution, whether or not the capsule isin a magnetic field or electrical field. The net charge is part of thephysical or chemical properties of the capsule at all times when in thesolution, and the net charge may be changed by the chemical nature ofthe solution or reagents expelled in certain embodiments. For example, apH change in the liquid phase of the solution or the liquid phase of thereagent can change or maintain the net charge of the capsule. Thecapsule, once disrupted, can maintain the same net charge as prior todisruption, or can have its charge changed after disruption. This isadvantageous when placing the capsule under an electrical current ormagnetic current and having a subsequent different result to the effectsof the current on the capsule based on the initial net charge of thecapsule before disruption or after disruption.

The encapsulation material used herein to make the encapsulated reagentsmay comprise micro-iron particles, or may be coated with an inertplastic or polymeric material.

The micro-iron particles of the encapsulation material can be any ferrocontaining particle (Fe) or other metal particles that can be moved by amagnet which is known and contemplated. The particle can be of the sizeless than or equal to 1×10⁻¹⁰, 1×10⁻⁹, 1×10⁻⁸, 1×10⁻⁷, 1×10⁻⁶, or 1×10⁻⁵meters.

The micro-iron particles (“micro particles”) can be coated with aceramic, plastic or polymeric coating to help in the stability of theparticles in solutions. The coating can be Teflon® or otherfluropolymer, for example. The micro particle can be by itself in thecapsule, in the reagent diluent or attached to a reagent in the capsule.The micro particle can be soluble, or at least partially soluble, orcolloidal in the diluent solution. If the micro particle is not attachedto a reagent element it could be used to mix or agitate the surroundingsolution. If the micro particle is attached to the capsule or reagent inthe capsule it can be used for mixing, agitating, or moving the reagent.In an alternative embodiment the diluent can have present therein anelectrolyte present to produce a net charge of the reagent present andto further the effectiveness of the magnet on the reagent.

Magnets that can be used in the present invention may be permanentmagnets, superconducting magnets, or resistive magnets, for example. Thepreferred embodiment is the use of a permanent magnet that has hightemperature stability for the use in high pressure and high temperatureconditions which may be used during in situ hybridization. Hightemperature stable permanent magnets which may be used herein aredescribed, for example, in U.S. Pat. No. 6,451,132 which is herebyexpressly incorporated herein by reference in its entirety. These hightemperature permanent magnets can be subjected to temperatures exceeding700° C. The magnets used herein may have Tesla ratings of 0.00001 Teslato 60 Tesla for example (one Tesla equals 10,000 Gauss). Preferably theGauss rating can be 1 to 20,000 Gauss.

Other magnets may be used such as Neodymium magnets which are a type ofpermanent magnet that can have the ability to retain its magneticproperties even under very high temperature conditions.

Most permanent magnets lose their magnetic properties when they areexposed to high heat conditions. A type of permanent magnet contemplatedfor the present invention has the grade of N42SH, the “SH” grade ofNeodymium permanent magnets can be used at temperatures over 150° C.Standard “N” grade permanents magnets have a maximum operatingtemperature of 80° C. A “SH” grade Neodymium permanent magnet with thedimensions of 2 inches long by 1 inch wide by one eighth inch has aGauss rating of 3095 for its surface field strength. It also has a Brmaxof 13,200 Gauss and a BHmax of 42 MGOe.

EXAMPLES Example 1: Encapsulated IHC Fast Red Chromogen Reagents

Fast Red chromogen protocols can be utilized in one embodiment of thepresent invention. It is well known in the art of alkaline phosphataseimmunohistochemistry (IHC) reactions that a final step of the protocolis to visualize the reaction by a color change at the antigen targetsite by the use of an activated fast red substrate/chromogen solutionthat reacts with the alkaline phosphatase enzyme attached to thebiotinylated antibody/streptavidin alkaline phosphatase complex. In theprior art method, chromogen activation is accomplished by adding 2milliliters of a substrate reagent solution (naphthol-phosphate in atris buffer) and 30-50 microliters of fast red chromogen reagentsolution together to form an activated chromogen. The activatedchromogen (i.e., the naphthol/tris/fast red chromogen complex) is a timedependent mixture that has a lifespan of only 30 minutes, after whichthe chromogen solution becomes inactive and must be rinsed off theslide. It is well known in the art of fast red chromogen chemistry thatfast red chromogen is not stable at room temperature, however, the fastred reagent is stable up to one year if stored under 2° C.-8° C.refrigeration. Fast red reagents can even degrade during shipment of thereagent from the manufacture to the end user. In the present invention,microencapsulated or nanoencapsulated chromagen present in a carriersolution can be used to make the “two part” fast red reagent system ofthe prior art into a single temperature stable solution that has bothreagents present in a single solution and that is stable at roomtemperature and doesn't have to be refrigerated during shipping. In aparticular advantageous embodiment of the present invention, the carriersolution is the naphthol-phosphate/buffer reagent, and the fast redreagent is encapsulated separately therein. When the time comes toexpose the fast red chromogen to the biological specimen, the techniciancan add the novel single fast red/carrier solution to the biologicalspecimen. The encapsulated reagents are disrupted by one of the methodsdescribed elsewhere herein. When the encapsulated fast red chromagenreagents are free to mix together, the fast red chromogen becomesactivated and can now form a color change when it comes in contact withthe alkaline phosphatase enzyme. This single solution can be anycombination of the required encapsulated reagents to render the singlesolution stable at room temperature as well as at a refrigeratedcondition. One can envision there would be a multitude of combinationsof encapsulated reagents, in the carrier solution, to product a stablesingle solution fast red chromogen reagent. In one example of thepresent invention, the fast red chromogen single solution comprisesdeionized water as the carrier solution and the remaining requiredreagents can be encapsulated separately or together or any combinationof a single reagent type per encapsulation or any combination ofreagents in the same encapsulation which can be disrupted to form anactivate fast red chromogen reagent which would be activated and readyfor use. In another embodiment, the deionized can have the tris bufferand naphthol present in its solution, and only the fast red chromogenreagent is encapsulated. In a preferred embodiment, the carrier solutionis comprised of deionized water, naphthol phosphate, and Tris buffer.The fast red chromogen reagent comprises a desiccated, lyophilized, dry,or powdered state and encapsulated. This solution is very stable at roomtemperatures, refrigerated temperatures, and shipping temperatures. Inone example of its use, this fast red chromogen-encapsulated solution isplaced onto the biological specimen and the solution is subject to apressurized environment (e.g., 0.01 psig to 5000 psig, preferably 1-200psig) which disrupts the desiccated, lyophilized, dry, or powderedencapsulated fast red reagent. The fast red reagent is quicklyreconstituted by the carrier solution and further reacts with thecarrier solution's reagents to form an activated fast red chromogen ableto react with the biological specimen's labeled antigen. This method ofuse can be performed using the methods and apparatuses of published U.S.patent applications 2006/0281116, 2006/0275889, and 2006/0275861, forexample. Alternatively, the disruption of the capsule can also be causedby any of the disruption modes listed elsewhere in this specification.The present invention is also contemplated for use with other chromogenssuch as horseradish peroxidase chromogens. The chemicals in this caseinclude hydrogen peroxide, deionized water, buffer, and DAB(3,3′-diaminobenzidine). The present invention also contemplates thatany combination of two or more reagents can be encapsulated (e.g., 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more),either together (if compatible) or separately encapsulated in a carriersolution. The present invention thus contemplates there can be at leastone or more reagents encapsulated and present in a carrier solution. Anynumber of combinations of encapsulated reagents and carrier solutionreagents is contemplated. A list of reagents which can be encapsulatedor present in the carrier solution is provided elsewhere herein. Thislist is for example only, is not exhaustive, and does not constitute anylimitation of the possible combinations of encapsulated reagent andreagent present in the carrier solution.

Example 2: Encapsulated DAB Chromogen Reagents

As noted the present invention can be used with other chromogens such ashorseradish peroxidase chromogens. The chemicals used in this embodimentare hydrogen peroxide, deionized water, buffer, and DAB(3,3′-diaminobenzidine), and DAB enhancers. In one example, the carriersolution comprises the buffer and hydrogen peroxide, and DAB can be theencapsulated reagent in the carrier solution. What in the prior art wasa three part, non-waiting system (buffer/DAB/hydrogen peroxide)necessary to obtain an activated chromogen is now, with the presentinvention, a one-step, one solution DAB chromogen. In this embodiment ofthe present invention, the encapsulated DAB is released under disruptionconditions to combine with the buffer and hydrogen peroxide to activatethe DAB chromogen for use. There can be multiple encapsulated reagentsthat are disrupted by different disruption modes, an example of which isthe last step of DAB chromogen, wherein the DAB chromogen attached tothe biological specimen is “enhanced” with a copper sulfate for changingthe color of DAB form brown to a darker brown or black in color. Theencapsulated copper sulfate in the carrier solution can be disrupted andreleased into the carrier solution, at the last step of the reaction, byany of the modes listed. Now what once was a 4 part system (in the priorart method) to activate DAB and enhance its color is now a singlesolution of the present invention that has two chemicals (DAB and Coppersulfate) encapsulated that can be released under a different disruptionmodes at different times in the reaction. It is obvious that thecombination of carrier solution and encapsulated reagent can be changed.For example the buffer and DAB could be free in the carrier solution andthe hydrogen peroxide could be the encapsulated component which isreleased under disruption to activate the chromogen for use.

Example 3: Encapsulated Pre-Treatment Enzymes

Any of the known enzymes that are used to digest the biologicalspecimens such as pepsin, ficin, and proteases can be encapsulated foruse as contemplated in the present invention. These proteins can beunstable in storage and during conventional use. The present inventioncontemplates encapsulated proteins in an aqueous carrier bufferedsolution and wherein when the disruption of choice occurs, the enzyme isreleased and able to react with the biological specimen. The presentinvention protects the enzyme from degradation during storage. Theseenzymes now can be stored at room temperature if desired. Themicroencapsulation protects the enzyme from degradation.

Example 4: Encapsulated Antibodies

Any of the known antibodies that are used to attach to biologicalspecimens during staining and/or antigen retrieval are contemplated foruse herein. Presently, these antibodies can be unstable in storage andduring use. The present invention can encapsulate these proteins in anaqueous carrier buffered solution and when the disruption of choiceoccurs, the antibody (or antibodies) is released and able to react withthe biological specimen. The present invention protects the antibodyfrom degradation during storage. These antibodies now can be stored atroom temperature if desired. The microencapsulation protects theantibodies from degradation. Antibodies of the present invention can beencapsulated separately (one type of antibody per encapsulation} ortogether to release several antibodies to react with the biologicalspecimen. (Several different types of antibodies in one capsule). Afurther possibility is each different antibody can be encapsulated in adifferent capsule separate from the rest which can be disrupted by thesame disruption condition or by different disruption conditions. Thisembodiment is advantageous when performing double stains and triplestain with antibodies reacting with a biological specimen.

In an example of a triple stain, a single solution can have threedifferent primary antibodies present in the carrier solution. Eachdifferent type of primary antibody is encapsulated into a separate typeof capsule having a different type of disruption mode. In the method ofits use, the first primarily antibody is disrupted and released andreacts with the biological specimen. This first primary antibody is thenreacted with a detection system (e.g., a secondary biotinylatedantibody/streptavidin label/chromogen) known in the art. The secondprimary antibody is then released by a different disruption mode andthen detected. The third primary antibody is released by a differentdisruption mode and then detected. The single solution can even havemultiple detection chemistries present to detect the primary antibodiespresent in the carrier solution. Any combination of primary antibodies,detection reagents, chromogens, buffers, etc. can be encapsulated in acarrier solution in accordance with the present invention. Theantibodies are protected from degeneration by being encapsulated. Theseprimary antibodies and their corresponding detection chemicals can bestored at room temperature or can be refrigerated. The preferredembodiment is storage of these reagents at room temperature. Theencapsulation inhibits the degradation of these proteins (antibodies)and chemicals from bacterial or fungal attack or physical degradation.The capsules may have anti-fungal and anti-bacterial properties toinhibit the degradation of these proteins and chemicals when stored atroom temperature or at refrigerated conditions.

Preferably the encapsulated reagent(s) in the carrier solutions of thepresent invention are stable under storage and/or shipping conditionswherein the carrier solutions with the reagents therein are exposed totemperature levels at low, refrigerated temperatures (<20° C.), roomtemperature (20° C.-25° C.), or temperatures above room temperature(>25° C.). The stability of the encapsulated reagents of the presentinventions as well as the decreased processing time or steps to activateor use reagents are primary advantages of the present invention. Theability to store the reagents of the present invention at roomtemperature is a key feature and advantage of the present invention.

In one embodiment, the encapsulated reagents and carrier solutions ofthe present invention are used in the reagent dispensing packs andstrips disclosed in U.S. Pat. Nos. 6,534,008, 6,855,292, 7,250,301,7,476,363, 7,622,077, 7,632,467, and Published Application numbers2006/0281116, 2006/0275889, and 2006/0275861 and pending U.S.application Ser. No. 12/550,288 each of which is expressly incorporatedherein by reference in its entirety. The advantage of the use of thereagents and solutions of the present invention is that reagentdispensing packs and reagent dispensing strips using these reagents andcarrier solutions can be shipped and stored at room temperature, asnoted above and can be stored at room temperature. This advantage isnovel because the technicians using staining apparatuses of the priorart must move their auto stainer reagents in and out of refrigeratorsthroughout the day to load and unload their auto stainers with requiredreagents. This refrigeration and heating up to room temperature of thereagents shortens the life of the reagents. Further, since thesereagents of the prior art require refrigeration there is generally aneed to have more than one refrigerator to store all the differentreagents. The reagent dispensing packs and strips which contains thereagents and carrier solutions of the present invention can be stored atroom temperature in a drawer, cabinet, or on the counter next to theauto staining instrument for example. The embodiment of the presentinvention makes storage of testing reagents more stable and reduces thetime for preparation and use. The present invention addresses the needfor room temperature storage of reagents used in medical and laboratorytesting, increased stability of reagents at room temperature andrefrigerated conditions, decreases in the processing time to “prepare”or “activate” reagents, and reduction of steps in the use of autostaining instruments.

As noted above, the carrier solutions described herein, which haveencapsulated reagents therein, may be supplied in or with or packaged insingle-use or multi-use reagent containers, packs or strips for use inmicroscope slide staining and antigen retrieval processes andapparatuses such as, but not limited to, those described in U.S. Pat.Nos. 6,534,008; 7,250,301; 6,855,292; 7,622,077; 7,632,461 and7,476,362, and US Published application 2006/0275889, and Pending U.S.application Ser. No. 12/550,296, each of which is expressly incorporatedherein by reference in its entirety.

The present invention is not to be limited in scope by the specificembodiments and examples described herein, since such embodiments andexamples are intended as but individual illustrations of one aspect ofthe invention and any similar or functionally equivalent embodiments arewithin the scope of this invention. Indeed, various modifications of thecompositions and methods of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art formthe foregoing description. Changes may be made in the construction andthe operation of the various components, compositions, elements,methods, and assemblies described herein or in the steps or the sequenceof steps of the methods described herein without departing from thespirit and scope of the invention as defined in the following claims.

Each of the references, patents or publications cited herein isexpressly incorporated herein by reference in its entirety.

What is claimed is:
 1. A solution in combination with an analyticsubstrate having a biological specimen treated with a horseradishperoxidase staining protocol disposed thereon, the solution comprising:a carrier solution disposed on the analytic substrate, the carriersolution comprising a chromogen activating buffer; and a plurality ofcapsules dispersed in the carrier solution, the capsules encapsulatingat least one reagent comprising a chromogen, wherein the solution ispositioned on the analytic substrate such that disruption of thecapsules causes the release of the reagent into the carrier solution sothat the reagent is activated by the chromogen activating buffer andbecomes available to act on or react with a horseradish peroxidase ofthe horseradish peroxidase staining protocol attached to or associatedwith the biological specimen.
 2. The solution of claim 1, wherein thecarrier solution is an oil based carrier solution.
 3. The combination ofclaim 1, wherein the carrier solution is an aqueous based carriersolution.
 4. The solution of claim 1, wherein the carrier solution is anon-aqueous based carrier solution.
 5. The solution of claim 1, whereinthe reagent is an oil based reagent.
 6. The solution of claim 1, whereinthe reagent is an aqueous based reagent.
 7. The solution of claim 1,wherein the reagent is a non-aqueous based reagent.
 8. The combinationof claim 1, wherein the reagent is a liquid.
 9. The solution of claim 1,wherein the reagent is dry.
 10. The solution of claim 1, wherein thereagent is a gel.
 11. The solution of claim 1, wherein the reagent is acolloidal.
 12. The solution of claim 1, wherein the reagent is anemulsion.
 13. The solution of claim 1, wherein the reagent islyophilized.
 14. The solution of claim 1, wherein the plurality ofcapsules further encapsulate at least one detergent.